PROJECT SUMMARY Diffusely infiltrative gliomas are the most common primary cancers in adults. Despite extensive research, these tumors are still incurable and lack effective treatment options. A subset of cells within gliomas, called glioma stem cells (GSC), exhibit a self-renewing stem-like phenotype, are highly tumorigenic, and are resistant to standard therapies. However, gliomas with mutations in isocitrate dehydrogenase 1 (IDH1mut) are much less aggressive than IDH1 wild-type (IDH1wt) gliomas that lack this mutation. IDH1mut causes global genomic hypermethylation, yet it is still unclear why IDH1mut gliomas are less aggressive, and how methylation of specific genes contributes to this unique phenotype, including whether hypermethylation impairs GSC development. We recently discovered that Tissue Factor (TF), a procoagulant that also promotes malignant behavior in cancers, is markedly hypermethylated and downregulated in IDH1mut gliomas compared to IDH1wt tumors. TF directly enhances the aggressiveness of cancer by signaling through G protein-coupled protease-activated receptor 2 (PAR2), but its role in gliomas is not well understood. Our preliminary data indicate that TF knockdown has multiple effects in IDH1wt gliomas, including (i) reduced activation of a critical oncogene in gliomas, epidermal growth factor receptor (EGFR); (ii) nearly complete elimination of multiple GSC markers; (iii) greatly impaired tumor malignancy. In contrast, PAR2 stimulation with a peptide agonist reverses the effects of TF knockdown. Furthermore, a new small molecule inhibitor of PAR2, GB83, greatly reduces IDH1wt glioma growth in vitro and is well-tolerated in mice. Because directly blocking TF risks disruption of hemostasis, whereas PAR2 is not required for TF to participate in normal clotting, GB83 could be an attractive new therapy against gliomas. Therefore, the objectives of this proposal are as follows: (i) determine the basis for reduced TF expression in IDH1mut gliomas; (ii) determine how intracellular TF-PAR2 signaling promotes GSC development and glioma malignancy; (iii) evaluate the in vivo therapeutic potential of PAR2 inhibition against gliomas with high TF-PAR2 activity. Using our unique panel of patient-derived xenografts (PDXs), we will test these lines of inquiry with three Aims. In Aim 1, we will determine whether promoter hypermethylation of the gene encoding TF, F3, is the reason why IDH1mut gliomas express very little TF. In Aim 2, we will determine how PAR2 activates EGFR, and whether this is the mechanism through which the GSC phenotype is induced by PAR2. In Aim 3, we will determine the therapeutic potential of PAR2 inhibition in gliomas. In sum, this research will increase our understanding of the molecular basis of the unique IDH1mut glioma phenotype, expand our understanding of PAR2 signaling pathways in glioma malignancy, and advance a new therapeutic strategy against gliomas.